Cyclosporin analogs for the treatment of lung diseases

ABSTRACT

the present invention relates to a cyclosporin analog of the following formula (I) or a pro-drug or pharmaceutically acceptable salt thereof:  
                 
 
     In formula I, the formula for residue A is:  
                 
 
     where X is absent, —C1-C6 alkyl-, or —C3-C6 cycloalkyl-; Y is selected from the groups: —C(O)—O—R1; —C(O)—S—R1; —C(O)—OCH2-OC(O)R2; —C(S)—O—R1; and —C(S)—S—R1; where R1 is hydrogen, C1-C6 alkyl optionally substituted with halogen, heterocyclics, aryl, C1-C6 alkoxy or C1-C6 alkylthio or halogen substituted C1-C6 alkoxy, halogen substituted C1-C6 alkylthio and where R2 is C1-C6 alkyl optionally substituted with halogen, C1-C6 alkoxy, C1-C6 alkylthio heterocyclics or aryl; B is -αAbu-, -Val-, -Thr- or -Nva-; and U is -(D)Ala-, -(D)Ser- or -[O-(2-hydroxyethyl)(D)Ser]-, or -[O-acyl(D)Ser]- or -[O-(2-acyloxyethyl)(D)Ser]-. In a second embodiment, the present invention relates to the use of the cyclosporin analogs of the present invention or a pro-drug or pharmaceutically acceptable salt thereof in pharmaceutical compositions for the treatment of asthma and other diseases characterized by airflow obstruction in a subject. In a third embodiment, the present invention relates to processes for the production of novel cyclosporin analogs of the present invention. The present invention also contemplates method(s) of treatment of asthma and other diseases characterized by airflow obstruction in a subject by administering to the subject therapeutically effective amounts of the cyclosporin analogs of the present invention with or without the concurrent use of other drugs or pharmaceutically acceptable carriers or excipients.

TECHNICAL FIELD

[0001] The present invention relates to novel cyclosporin analogs andmethods for the treatment of asthma and other diseases characterized byairflow obstruction in a subject. The present invention further relatesto pharmaceutical compositions comprising the compounds of the presentinvention and processes for their production.

BACKGROUND OF THE INVENTION

[0002] Respiratory diseases, such as asthma and other diseasescharacterized by airflow obstruction, are a global problem. Millions ofpeople worldwide, both children and adults, suffer from these medicalconditions. These diseases reduce quality of life by impairing theability of sufferers to perform everyday tasks, and in some cases, causedeath. One of the major respiratory diseases is asthma.

[0003] Asthma is a disease of unknown etiology in which the bronchi areinflamed and as a consequence obstructed. This narrowing results from acombination of bronchial smooth muscle contraction, mucosal oedema,inflammatory cell infiltrate and partial or total occlusion of the lumenwith mucus, cells and cell debris. Bronchial obstruction is eitherpartially or totally reversible, and this important featuredistinguishes asthma from chronic bronchitis.

[0004] Asthma is an extremely common disease with a worldwide prevalenceof 5% to 8%. In the developed world it is the most common chronicillness and, for reasons that are unclear, the disease is on theincrease. It is now accepted that asthma is a chronic inflammatorydisorder of the airways in which many cells play a role, in particular,mast cells, eosinophils and T-lymphocytes. In susceptible individualsthis inflammation causes symptoms which are usually associated withwidespread but variable airflow obstruction. This type of airflowobstruction is often reversible either spontaneously or with treatmentand causes associated increase in airway responsiveness to a variety ofstimuli.

[0005] The illness has a wide clinical spectrum ranging from mildepisodic bronchospasm (easily controlled by the occasional use of abronchodilator) to a very severe intractable asthma sometimes resistantto treatment with high doses of oral corticosteroids. Steroid resistanceoccurs in less than 5% of people with asthma. This translates tothousands of people. These patients with severe chronic disease may bedependent on corticosteroids and their disease is often so severe thatfull reversibility can be difficult or impossible to demonstrate.

[0006] Chronic obstructive airways disease, chronic obstruction lungdisease and ‘smoker's chest’ have all been used to describe what is nowknown as COPD. COPD is characterized by progressive irreversible airwayobstruction. It can lead to death from respiratory or cardio-respiratoryfailure. COPD consists of two subsets: chronic bronchitis and emphysema.In practice, it is very difficult to define the contribution of each ofthese two conditions to the obstruction of the airway and this has ledto the displacement of these labels by the non-specific term COPD. Thepathology of COPD is not fully elucidated, but features includehypertrophy of mucus-secreting glands, inflammation (includinginfiltration with lymphocytes) and goblet cell hyperplasia.

[0007] The treatment of COPD consists of bronchodilators, intermittentcourses of antibiotics and, in some patients, inhaled and/or oralcorticosteroids. The latter is claimed to reduce the decline in lungfunction in COPD.

[0008] Cystic fibrosis is an inherited condition. Excess viscid mucus isproduced. This leads to recurrent chest infections and progressivebronchiectasis. Approximately 50% of cystic fibrosis sufferers havebronchial hyperresponsiveness and there is an increased incidence ofatopy. There is widespread airway narrowing and wheeze. Most cysticfibrosis sufferers take bronchodilators, some take inhaledcorticosteroids. And at least one study had reported benefit with oralcorticosteroids.

[0009] Current drugs for treating asthma are corticosteroids (such asbeclomethasone, triamcinolone), beta adrenergics (such as epinephrine,albuterol, bitolterol), NSAIDS, leukotriene antagonists, Xanthines(methyl xanthines such as theophylline, oxtriphylline) andanticholinergics (such as atropine, ipratropium bromide).

[0010] Corticosteroids are the mainstay of treatment of chronic asthmaand they revolutionized the treatment of this disease when they werefirst introduced in the 1950's. Oral corticosteroids have today beenlargely replaced by inhaled corticosteroids, although severe asthmaticsstill require medication by mouth. Inhaled cortisteroids are relativelysafe and extremely effective in most patients, and improved the qualityof life for millions of asthmatic sufferers. For those with severeasthma, however, oral therapy with corticosteroids is required. Whentaken for more than a few days oral corticosteroids have a number ofserious side effects. These include growth retardation in children,severe osteoporosis (especially in old age), decreased responsiveness ofthe pituitary adrenal axis to stress, fluid retention, diabetes andprecipitation of psychosis.

[0011] Furthermore, an appreciable number of patients have apparentcorticosteroid resistance or unreponsiveness. Patients consideredsuccessfully treated with inhaled or oral steroids often have to becontent with 60% of their predicted lung function. Further increasingthe dose of oral corticosteroids runs the risk of concomitant sideeffects.

[0012] Although corticosteroids are effective for asthma, they are notideal drugs. Over the years doctors have occasionally usedimmunosuppressive agents as adjuncts to corticosteroids in patients withextremely severe disease. Examples of immunosuppressive drugs areazathioprine, methotrexate, mycophenolic acid and prodrug, leflunomide,Cyclosporin A, ascomycin, FK-506 and rapamycin.

[0013] The cyclosporins comprise a class of structurally distinctive,cyclic, poly-N-methylated undecapeptides, commonly possessingpharmacological activity, in particular immunosuppressive,anti-inflammatory or anti-parasitic activity. The first of thecyclosporins to be isolated was the naturally occurring fungalmetabolite cyclosporin, Cyclosporin A represented as follows:

[0014] Since the original discovery of cyclosporin, a wide variety ofnaturally occurring cyclosporins have been isolated and identified, andmany further non-natural cyclosporins have been prepared by total- orsemi-synthetic means or by the application of modified culturetechniques. The class comprising cyclosporins is thus now substantialand includes, for example, the naturally occurring Cyclosporins Athrough Z, for example, [Thr]², [Val]², [Nva]² and [Nva]²⁻, [Nva]⁵-Cyclosporin (also known as Cyclosporins C, D, G and M respectively),[(D)MeVal]¹¹-Cyclosporin (also known as Cyclosporin H), [cf., Traber etal.;1, Helv. Chim. Acta, 60,1247-1255 (1977); Traber et al.; 2, Helv.Chim. Acta, 65, 1655-1667 (1982); Kobel et al.; Europ. J. AppliedMicrobiology and Biotechnology, 14, 273-240 1982); and Von Wartburg etal.; Progress in Allergy, 38, 28-45,1986)]; as well as variousnon-natural cyclosporin derivatives and artificial or syntheticcyclosporin derivatives and artificial or synthetic cyclosporinsincluding dihydrocyclosporins [in which the MeBmt-residue is saturatedby hydrogenation]; derivatized cyclosporins (e.g., in which the3′-O-atom of the MeBmt-residue is acylated or a further substituent isintroduced at the α-carbon atom of the sarcosyl residue at the3-position); and cyclosporins in which variant amino acids areincorporated at specific positions within the peptide sequence, forexample, [3-O-acetyl-MeBmt]¹-Cyclosporin (also known asDihydro-cyclosporin D), [(D)Ser]⁸-Cyclosporin, [Melle]¹¹-Cyclosporin,[MeAla]⁶-Cyclosporin, [(D) Pro]³-Cyclosporin etc., employing the totalsynthetic method for the production of cyclosporins developed by R.Wenger—see e.g. Traber et al., 1; Traber et al., 2; and Kobel et al.,loc cit. U.S. Pat. Nos. 4,108,985, 4,220,641, 4,288,431, 4,554,351,4,396,542 and 4,798,823; European Patent Publication Nos. 34,567A,56,782A, 300,784A and 300,785; International Patent Publication No. WO86/02080 and UK Patent Publication Nos. 2,206,119 and 2,207,678; Wenger1, Transpl. Proc., 15 Suppl. 1:2230 (1983); Wenger 2, Angew. Chem. Int.Ed. 24 77 (1985) and Wenger 3, Progress in the Chemistry of OrganicNatural Products, 50, 123 (1986).

[0015] There is increasing evidence that chronic inflammation in asthmais mediated via a network of cytokines emanating from inflammatory andstructural cells in the airways. The prominent eosinophilic inflammationthat characterizes asthma appears to be orchestrated by cytokinesderived from type 2 T-helper (Th2)-like lymphocytes, suggesting thatimmunosuppressants might be beneficial in the control of asthma (see forexample, “Pharmacokinetics, pharmacodynamics, and safety of inhaledcyclosporin A after single and repeated administration in ,healthy maleand female subjects and asthmatic patients,” Rohatagi, S. et al.,Aventis Pharmaceutical, Collegeville, Pa., USA. J. Clin. Pharmacol.(2000), 40(11), 1211-1226). Cyclosporin A (hereinafter “CsA”) is activeagainst CD4+ lymphocytes and might, therefore, be useful for asthma. Atrial of low-dose oral CsA in patients with steroid-resistant asthmaindicated that it can improve control of symptoms in patients withsevere asthma on oral steroids.

[0016] The mechanism of CsA action in asthma is of interest. CsA bindsto the ubiquitous protein cyclophilin, in the cytosol, and the complexin turn binds to calcineurin, which is a calcium and calmodulindependent serine threonine phosphatase. Calcineurin is necessary for thecytoplasmic portion of the transcription factor NF-AT, a nuclear factorof activated T-cells, to translocate to the nucleus and bind to itsnuclear portion to become an active transcription factor. NF-AT forms acomplex with AP-1 and regulates the transcription of the IL-2 gene,together with other genes, for example, IL-5. CsA prevents thecytoplasmic portion of NF-AT from translocating, resulting in reducedtranscription of IL-2. CsA has a specific inhibitory effect in CD4+cells through this transcription mechanism, but may also have inhibitoryeffects on other cells, including mast cells and eosinophils, throughmechanisms that have not yet been defined.

[0017] Recently, three controlled trials of CsA in asthma have beenreported. [Alexander A G, Barnes N C, Kay A B. Trial of cyclosporin incorticosteroid-dependent chronic severe asthma. Lancet 1992; 339:324-328; Niwanowska E, Dworski R, Domala B, Pinis G. Cyclosporin forsteroid-dependent asthma. Allergy, 1991; 46: 312-315; Lock S H, Kay A B,Barnes N C. Double-blinded, placebo-controlled study of cyclosporin A asa corticosteroid-sparing agent in corticosteroid-dependent asthma. Am JRespir Crit Care Med 1996; 153: 509-14; Nizankowska E, Soja J, Pinis G,Bochenek G, Sladek K, Domagala B, et al. Treatment of steroid-dependentbronchial asthma with cyclosporin. Eur Respir J 1995; 8: 1091-1099.]

[0018] CsA 5 mg/kg/day allowed a significant reduction in the use ofcorticosteroids by 60%. Side effects with systemic CsA were increase indiastolic blood pressure and decrease in renal function. Other sideeffects include hepatic dysfunction, hypertrichosis, tremor, gingivalhyperplasis and paraesthesia. The systemic toxicity of CsA limits itsuse for the treatment of asthma, COPD and other related lung diseases.Therefore, it is desirable to synthesize analogs of CsA which retainCsA's potential utility as a primary or adjunct therapy for respiratorydiseases, while reducing or eliminating CsA's systemic toxicity.

SUMMARY OF THE INVENTION

[0019] The present invention relates to novel cyclosporin analogs andmethods of treatment for the treatment of asthma and other diseasescharacterized by airflow obstruction in a subject. The present inventionfurther relates to pharmaceutical compositions comprising the compoundsof the present invention and processes for their production.

[0020] More particularly, the present invention relates to a cyclosporinanalog of the following formula (I) or a pro-drug or pharmaceuticallyacceptable salt thereof:

[0021] In formula I, the formula for residue A is:

[0022] where X is absent, —C1-C6 alkyl-, or —C3-C6 cycloalkyl-; Y isselected from the groups: —C(O)—O—R1; —C(O)—S—R1; —C(O)—OCH2-OC(O)R2;—C(S)—O—R1; and —C(S)—S—R1; where R1 is hydrogen, C1-C6 alkyl optionallysubstituted with halogen, heterocyclics, aryl, C1-C6 alkoxy or C1-C6alkylthio or halogen substituted C1-C6 alkoxy, halogen substituted C1-C6alkylthio and where R2 is C1-C6 alkyl optionally substituted withhalogen, C1-C6 alkoxy, C1-C6 alkylthio heterocyclics or aryl; B is-αAbu-, -Val-, -Thr- or -Nva-; and U is -(D)Ala-, -(D)Ser- or-[O-(2-hydroxyethyl)(D)Ser]-, or -[O-acyl(D)Ser]- or-[O-(2-acyloxyethyl)(D)Ser]-.

[0023] In a second embodiment, the present invention relates to the useof the cyclosporin analogs of the present invention or a pro-drug or apharmaceutically acceptable salt thereof in pharmaceutical compositionsfor the treatment of asthma and other diseases characterized by airflowobstruction in a subject.

[0024] In a third embodiment, the present invention relates to processesfor the production of novel cyclosporin analogs of the presentinvention. In a preferred embodiment, the present invention relates tothe processes for the production of cyclosporin analogs of formula I,with the structure of residue A as illustrated above.

[0025] The present invention also contemplates method(s) of treatment ofasthma and other diseases characterized by airflow obstruction in asubject by administering to the subject therapeutically effectiveamounts of the cyclosporin analogs of the present invention with orwithout the concurrent use of other drugs or pharmaceutically acceptablecarriers or excipients.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention relates to novel cyclosporin analogs andmethods of treatment for the treatment of asthma and other diseasescharacterized by airflow obstruction in a subject. The present inventionfurther relates to pharmaceutical compositions comprising the compoundsof the present invention and processes for their production. The patentsand publications identified in this specification indicate the knowledgein this field and are hereby incorporated by reference in theirentirety. In the case of inconsistencies, the present disclosure willprevail.

[0027] More particularly, the present invention relates to a cyclosporinanalog of the following formula (I) or a pro-drug or pharmaceuticallyacceptable salt thereof:

[0028] In formula I, the formula for residue A is:

[0029] where X is absent, —C1-C6 alkyl-, or —C3-C6 cycloalkyl-; Y isselected from the groups: —C(O)—O—R1 where R1 is hydrogen, C1-C6 alkyloptionally substituted with halogen, heterocyclics, aryl, C1-C6 alkoxyor C1-C6 alkylthio, halogen substituted C1-C6 alkoxy or halogensubstituted C1-C6 alkylthio; —C(O)—S—R1 where R1 is hydrogen, C1-C6alkyl optionally substituted with halogen, heterocyclics, aryl, C1-C6alkoxy or C1-C6 alkylthio, halogen substituted C1-C6 alkoxy or halogensubstituted C1-C6 alkylthio; —C(O)—OCH2-OC(O)R2 where R2 is C1-C6 alkyloptionally substituted with halogen, C1-C6 alkoxy, C1-C6 alkylthioheterocyclics or aryl; —C(S)—O—R1 where R1 is hydrogen, C1-C6 alkyloptionally substituted with halogen, heterocyclics, aryl, C1-C6 alkoxyor C1-C6 alkylthio, halogen substituted C1-C6 alkoxy or halogensubstituted C1-C6 alkylthio; and —C(S)—S—R1 where R1 is hydrogen, C1-C6alkyl optionally substituted with halogen, heterocyclics, aryl, C1-C6alkoxy or C1 -C6 alkylthio, halogen substituted C1-C6 alkoxy, halogensubstituted C1-C6 alkylthio; B is -αAbu-, -Val-, -Thr- or -Nva-; and Uis -(D)Ala-, -(D)Ser- or -[O-(2-hydroxyethyl)(D)Ser]-, or-[O-acyl(D)Ser]- or -[O-(2-acyloxyethyl)(D)Ser]-.

[0030] In formula I, abbreviation of amino acid residues, for example,-Ala-, -MeLeu-, -αAbu-, etc., are in accordance with conventionalpractice and are to be understood as having the L-configuration unlessotherwise indicated (for example, -(D)Ala- represents a residue havingthe D-configuration). Abbreviation of residues preceded by “Me-”represents a α-N-methylated amino acid residue, for example, “Me-Leu” isa α-N-methylated-Leucine residue. Individual residues of a molecule ofthe cyclosporin analog of the present invention are numbered, as in theart, clockwise and starting with the residue -MeBmt-, corresponding toresidue 1. The same numerical sequence is employed throughout thepresent specification and claims.

[0031] In a most preferred embodiment, a cyclosporin analog of thepresent invention is represented by formula I or a pro-drug orpharmaceutically acceptable salt thereof, where residue B is -αAbu- andresidue U is -(D)Ala-. In another preferred embodiment, the cyclosporinanalog of the present invention is represented by formula I or apro-drug or pharmaceutically acceptable salt thereof, where X is absentin residue A, residue B is -αAbu- and residue U is -(D)Ala-.

[0032] Representative compounds of the invention include, but are notlimited to, the following compounds as illustrated below:

[0033] Compound of formula I, where in residue A, X is absent andY=—COOCH₃; residue B=-αAbu-, and residue U=-(D)Ala-.

[0034] Compound of formula I, where in residue A, X is absent andY=—COOH; residue B=-αAbu-, and residue U=-(D)Ala-.

[0035] Compound of formula I, where in residue A, X is absent andY=-COOEt; residue B=-αAbu-, and residue U=-(D)Ala-.

[0036] Compound of formula I, where in residue A, X is absent andY=—COOCH₂CH₂CH₃; residue B=-αAbu-, and residue U=-(D)Ala-.

[0037] Compound of formula I, where in residue A, X is absent andY=—COOCH₂Ph; residue B=-αAbu-, and residue U=-(D)Ala-.

[0038] Compound of formula I, where in residue A, X is absent andY=—COOCH₂F; residue B=-αAbu-, and residue U=-(D)Ala-.

[0039] Compound of formula I, where in residue A, X is absent andY=—COOCHF₂; residue B=-αAbu-, and residue U=-(D)Ala-.

[0040] Compound of formula I, where in residue A, X is absent andY=—COOCF₃; residue B=-αAbu-, and residue U=-(D)Ala-.

[0041] Compound of formula I, where in residue A, X is absent andY=—COOCH₂CF₃; residue B=-αAbu-, and residue U=-(D)Ala-.

[0042] Compound of formula I, where in residue A, X is absent andY=—COOCH₂Cl; residue B=-αAbu-, and residue U=-(D)Ala-.

[0043] Compound of formula I, where in residue A, X is absent andY=—COOCH₂OCH₃; residue B=-αAbu-, and residue U=-(D)Ala-.

[0044] Compound of formula I, where in residue A, X is absent andY=—COOCH₂OCH₂CH₂OCH₃; residue B=-αAbu-, and residue U=-(D)Ala-.

[0045] Compound of formula I, where in residue A, X is absent andY=—C(═O)SCH₂Ph; residue B=-αAbu-, and residue U=-(D)Ala-.

[0046] Compound of formula I, where in residue A, X is —CH₂CH₂CH₂— andY=—COOCH₃; residue B=-αAbu-, and residue U=-(D)Ala-.

[0047] Compound of formula I, where in residue A, X is absent andY=—COOFmoc; residue B=-αAbu-, and residue U=-(D)Ala-.

[0048] Cyclosporin analogs of the invention are accordingly useful forthe treatment of diseases or conditions responsive to or requiringtopical anti-inflammatory, immunosuppressive or related therapy, forexample, topical administration for the treatment of such diseases orconditions of the eye, nasal passages, buccal cavity, skin, colon or,especially, airways or lung. In particular, cyclosporin analogs of theinvention permit topical anti-inflammatory, immunosuppressive or relatedtherapy with the concomitant avoidance or reduction of undesirablesystemic side effects, for example general systemic immunosuppression.

[0049] Cyclosporin analogs of the invention useful for the treatment ofdiseases and conditions of the airways or lung, in particular,inflammatory or obstructive airway diseases. They are especially usefulfor the treatment of diseases or conditions of the airways or lungsassociated with or characterized by inflammatory cell infiltration orother inflammatory events accompanied by inflammatory cell accumulation,for e.g., eosinophil and/or neutrophil. Most preferably, they are usefulfor the treatment of asthma.

[0050] Cyclosporin analogs of the invention are useful in the treatmentof asthma of whatever type of genesis including both intrinsic and,especially, extrinsic asthma. They are useful for the treatment ofatopic and non-atopic asthma, including allergic asthma, bronchiticasthma, exercise induced asthma, occupational asthma, asthma inducedfollowing bacterial infection and other non-allergic asthmas. Treatmentof asthma is also to be understood as embracing treatment of“wheezy-infant syndrome,” that is treatment of subjects, for example, ofless than 4 to 5 years of age, exhibiting wheezing symptoms, inparticular at night, and diagnosed or diagnosable as “wheezy infants,”an established patient category of major medical concern and now morecorrectly indentified as incipient or early-phase asthmatics.Cyclosporin analogs of the invention are in particular useful for thetreatment of asthma in subjects whose asthmatic status is either steroiddependent or steroid resistant.

[0051] Cyclosporin analogs of the invention are also useful for thetreatment of bronchitis or for the treatment of chronic or acute airwaysobstruction associated therewith. Cyclosporin analogs of the inventionmay be used for the treatment of bronchitis of whatever type or genesis,including, for example, acute bronchitis, arachidic bronchitis,catarrhal bronchitis, chronic bronchitis, croupous bronchitis, phthinoidbronchitis and so forth.

[0052] Cyclosporin analogs of the invention are in addition useful forthe treatment of pneumoconiosis (an inflammatory, commonly occupational,disease of the lungs, frequently accompanied by airways obstruction,whether chronic or acute, and occasioned by repeated inhalation ofdusts) of whatever type or genesis, including, for example, aluminosis,anthracosis, asbestosis, berylliosis, chalicosis, ptilosis, siderosis,silicosis, tabacosis and, in particular, byssinosis.

[0053] Cyclosporin analogs of the invention may also be used for thetreatment of eosinophil-related disorders of the airways (e.g. involvingmorbid eosinophilic infiltration of pulmonary-tissues) includinghypereosinophilia as it effects the airways and/or lungs as well as, forexample, eosinophil-related disorders of the airways consequential orconcomitant to Loffler's syndrome, eosinophilic pneumonia, parasitic (inparticular metazoan) infestation (including tropical eosinophilia),bronchopulmonary aspergillosis, polyarteritis nodosa (includingChurg-Strauss syndrome), eosinophilic granuloma and eosinophil-relateddisorders affecting the airways occasioned by drug-reaction.

[0054] The word “treatment” as used herein in relation to the treatmentof diseases of the airways and lungs, in particular asthma, is to beunderstood as embracing both symptomatic and prophylactic modes, that isfor immediate treatment, for e.g., of acute inflammation (symptomatictreatment) as well as for advance treatment to prevent, ameliorate orrestrict long term symptomatology (prophylactic treatment). The term“treatment” as used in the present specification and claims in relationto such diseases is to be interpreted accordingly as including bothsymptomatic and prophylactic treatment, for e.g., in the case of asthma,symptomatic treatment to ameliorate acute inflammatory events andprophylactic treatment to restrict on-going inflammatory status and toameliorate future bronchial exacerbation associated therewith.

[0055] Cyclosporin analogs of the invention may also be used to treatany disease or condition of the airways or lungs requiringimmunosuppressive therapy, for e.g., the treatment of autoimmunediseases, or as they affect, the lungs (for example, for the treatmentof sarcoidosis, alveolitis or chronic hypersensitivity pneumonitis) orfor the maintainance of allogenic lung transplant, for e.g., followinglung or heart lung transplantation.

[0056] As previously indicated, for the above purposes, cyclosporinanalogs of the invention will be administered topically within theairways, for e.g., by the pulmonary route or by inhalation. As alsopreviously noted, while having potent efficacy when administeredtopically; cyclosporin analogs of the invention exhibit reduced systemictoxicity. Cyclosporin analogs of the invention thus provide a means forthe treatment of diseases and conditions of the airways or lung, forexample, as hereinabove set forth, with the avoidance of unwantedsystemic side effect, e.g. consequent to inadvertent swallowing of drugsubstance during inhalation therapy. It is estimated that during thecourse of manoeuvres required to effect administration by inhalation, upto 90% or more of total drug substance administered will normally beswallowed rather than inhaled.

[0057] By the provision of cyclosporin analogs which are topicallyactive, e.g. effective when inhaled, but systemically inactive thepresent invention makes cyclosporin therapy available to subjects forwhom such therapy might otherwise be excluded, e.g. due to the risk ofsystemic, in particular immunosuppressive, side effect.

[0058] Further uses include the treatment and prophylaxis ofinflammatory and hyperproliferative skin diseases and cutaneousmanifestations of immunologically-mediated illnesses, such as psoriasis,atopical dermatitis, contact dermatitis and further eczematousdermatitises, seborrhoeis dermatitis, Lichen planus, Pemphigus, bullouspemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides,erythemas, cutaneous eosinophilias, Lupus erythematosus, acne andAlopecia areata; various eye diseases (autoimmune and otherwise) such askeratoconjunctivitis, vernal conjunctivitis, keratitis, herpetickeratitis, conical cornea, dystrophia epithelialis corneae, cornealleukoma, ocular pemphigus, Mooren's ulcer, Scleritis, Graves'opthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, multiplemyeloma, etc.; obstructive airway diseases, which includes conditionssuch as COPD asthma (for example, bronchial asthma, allergic asthma,intrinsic asthma, extrinsic asthma and dust asthma), particularlychronic or inveterate asthma (for example, late asthma and airwayhyper-responsiveness), bronchitis, allergic rhinitis and the like;inflammation of mucosa and blood vessels such as gastric ulcers,vascular damage caused by ischemic diseases and thrombosis. Moreover,hyperproliferative vascular diseases such as intimal smooth muscle cellhyperplasia, restenosis and vascular occlusion, particularly followingbiologically- or mechanically-mediated vascular injury can be treated orprevented by the compounds of the invention.

[0059] The compounds of the present invention may also find utility inthe chemosensitization of drug resistant target cells. Cyclosporin A andFK-506 are known to be effective modulators of P-glycoprotein, asubstance which binds to and inhibits the action of anticancer drugs; byinhibiting P-glycoprotein, they are capable of increasing thesensitivity of multidrug resistant (MDR) cells to chemotherapeuticagents. It is believed that the compounds of the invention may likewisebe effective at overcoming resistance expressed to clinically usefulantitumour drugs such as 5-fluorouracil, cisplatin, methotrexate,vincristine, vinblastine and adriamycin, colchicine and vincristine.

[0060] Accordingly, the pharmaceutical compositions of the presentinvention comprise a therapeutically effective amount of a cyclosporinanalog of the invention in combination with a pharmaceuticallyacceptable carrier or excipient. In particular, compositions pertainingto the present invention are useful for treating a subject for areversible obstructive airway disease.

[0061] The present invention also contemplates method(s) of treatment ofasthma and other diseases characterized by airflow obstruction in asubject by administering to the subject therapeutically effectiveamounts of the cyclosporin analogs of the present invention with orwithout the concurrent use of other drugs or pharmaceutically acceptablecarriers or excipients, as described throughout the presentspecification. Such treatment of the disease may be done byadministering a therapeutically effective amount of a compound of theinvention for such time and in such amounts as is necessary to producethe desired result.

[0062] As used in the present invention, “therapeutically effectiveamount” of one of the compounds means a sufficient amount of thecompound to treat a particular disease, at a reasonable benefit/riskratio. The compounds of the present invention may be employed in pureform or, where such forms exist, in pharmaceutically acceptable salt,ester or prodrug forms. Alternatively, the compound may be administeredas pharmaceutical compositions containing the compound of interest incombination with one or more drugs or pharmaceutically acceptableexcipients. It will be understood, however, that the total daily usageof the compounds and compositions of the present invention will bedecided by the attending physician within the scope of sound medicaljudgment.

[0063] The specific therapeutically-effective dose level for anyparticular patient will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of the compound at levelslower than required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved.

[0064] Dosages of the cyclosporin analogs of the present inventionemployed in practicing the method of the present invention will ofcourse vary depending on the site of treatment, the particular conditionto be treated, the severity of the condition, the subject to be treated(for e.g., in terms of body weight, age and so forth) as well as theeffect desired. In general, for treating diseases or conditions of theairways or lungs, for e.g., inflammatory or obstructive airway diseasesuch as asthma, cyclosporins of the invention can be suitablyadministered topically to the airways or lungs, for e.g., but notlimited to, inhalation, at dosages from about 20 to about 400 mg/day,preferably from about 50 to about 300 mg/day, most preferably from about200 to about 300 mg/day. Dosages will appropriately be administered froma metered delivery system in a series of from 1 to 5 puffs at eachadministration, with administration performed once to four times daily.Dosages at each administration will thus conveniently be from about 5 to100 mg/day, more preferably from about 12.5 to about 100 mg/day, e.g.administered with a metered delivery device capable of delivering, fore.g., 1 to 25 mg cyclosporin per actuation. For purposes of oraladministration, more preferable doses may be in the range from about0.005 to about 3 mg/kg/day. If desired, the effective daily dose may bedivided into multiple doses for purposes of administration;consequently, single dose compositions may contain such amounts orsubmultiples thereof to make up the daily dose.

[0065] Definitions

[0066] The terms “C₁-C₃-alkyl” or “C₁-C₆-alkyl” as used herein refer tosaturated, straight- or branched-chain hydrocarbon radicals containingbetween one and three or one and six carbon atoms, respectively.Examples of C₁-C₃ alkyl radicals include methyl, ethyl, propyl andisopropyl, and examples of C₁-C₆-alkyl radicals include, but are notlimited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl,neopentyl and n-hexyl.

[0067] The term “C₁-C₆alkoxy” as used herein refers to an C₁-C₆-alkylgroup, as previously defined, attached to the parent molecular moietythrough an oxygen atom. Examples of C₁-C₆-alkoxy include, but are notlimited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy,neopentoxy and n-hexoxy.

[0068] The term “C₁-C₆-alkylthio” as used herein refers to anC₁-C₆-alkyl group, as previously defined, attached to the parentmolecular moiety through a sulfur atom. Examples of C₁-C₆-alkylthioinclude, but are not limited to, thiomethoxy, thioethoxy, thiopropoxy,thio-isopropoxy, n-thiobutoxy, tert-thiobutoxy, neothiopentoxy andn-thio-hexoxy.

[0069] The term “aryl” as used herein refers to a carbocyclic ringsystem having one or more aromatic rings including, but not limited to,phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl and the like.Aryl groups (including multi-cyclic aryl groups) can be unsubstituted orsubstituted with one, two or three substituents independently selectedfrom lower alkyl, substituted loweralkyl, haloalkyl, alkoxy, thioalkoxy,lower alkylenedioxy, lower alkylidenedioxy, amino, alkylamino,dialkylamino, acyamino, cyano, hydroxy, acyl, halo and/ortrifluoromethyl, mercapto, nitro, carboxylaldehyde, carboxy,alkoxycarbonyl, carbamoyl, sulfamoyl, lower alkoxycarbonylamino, loweralkanoyl, ureido, amidino and carboxamide. In addition, substituted arylgroups include tetrafluorophenyl and pentafluorophenyl.

[0070] The term “C₃-C₆-cycloalkyl-” as used herein refers to carbocyclicgroups of 3 to 6 carbons, respectively; for example, cyclopropyl,cyclobutyl, cyclopentyl and cyclohexyl.

[0071] The terms “halo” and “halogen” as used herein refer to an atomselected from fluorine, chlorine, bromine and iodine.

[0072] The term “heterocyclics”, as used herein, refers to a cyclicaromatic radical having from five to ten ring atoms of which one ringatom is selected from S, O and N; zero, one or two ring atoms areadditional heteroatoms independently selected from S, O and N; and theremaining ring atoms are carbon, the radical being joined to the rest ofthe molecule via any of the ring atoms, such as, for example, pyridinyl,pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl,oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl,quinolinyl, isoquinolinyl, and the like.

[0073] The term “subject” as used herein refers to a mammal or animal.Preferably the mammal is a human. A subject refers to, for example,dogs, cats, horses, cows, pigs, guinea pigs and the like.

[0074] The term “pro-drug” as used herein refers to pharmacologicallyacceptable derivatives, for example, but not limited to, esters andamides, such that the resulting biotransformation product of thederivative is the active drug. Pro-drugs are known in the art and aredescribed generally in, e.g., Goodman and Gilman's “Biotransformation ofDrugs,” in the Pharmacological Basis of Therapeutics, 8^(th) Ed., McGrawHill, Int. Ed. 1992, page 13-15, which is hereby incorporated byreference in its entirety.

[0075] As used herein, the term “pharmaceutically acceptable salt”refers to those salts which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andlower animals without undue toxicity, irritation, allergic response andthe like, and are commensurate with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge, et al. describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporatedherein by reference. The salts can be prepared in situ during the finalisolation and purification of the compounds of the invention, orseparately by reacting the free base function with a suitable organicacid. Examples of pharmaceutically acceptable, nontoxic acid additionsalts are salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, ptoluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

[0076] Pharmaceutical Compositions

[0077] The pharmaceutical compositions of the present invention comprisea therapeutically effective amount of a compound of the presentinvention formulated together with one or more pharmaceuticallyacceptable carriers. As used herein, the term “pharmaceuticallyacceptable carrier” means a non-toxic, inert solid, semi-solid or liquidfiller, diluent, encapsulating material or formulation auxiliary of anytype. Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as lactose, glucose and sucrose;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil; safflower oil; sesame oil; olive oil; corn oil andsoybean oil; glycols; such a propylene glycol; esters such as ethyloleate and ethyl laurate; agar; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe composition, according to the judgement of the formulator. Thepharmaceutical compositions of this invention can be administered tohumans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, or as an oral or nasal spray.

[0078] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

[0079] Solid dosage forms for oral administration include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound is mixed with at least one inert, pharmaceuticallyacceptable excipient or carrier such as sodium citrate or dicalciumphosphate and/or a) fillers or extenders such as starches, lactose,sucrose, glucose, mannitol, and silicic acid, b) binders such as, forexample, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such asglycerol, d) disintegrating agents such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate, e) solution retarding agents such as paraffin, f) absorptionaccelerators such as quaternary ammonium compounds, g) wetting agentssuch as, for example, cetyl alcohol and glycerol monostearate, h)absorbents such as kaolin and bentonite clay, and i) lubricants such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof. In the case of capsules,tablets and pills, the dosage form may also comprise buffering agents.

[0080] Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugar as well as high molecular weight polyethyleneglycols and the like.

[0081] The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

[0082] Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugar as well as high molecular weight polethyleneglycols and the like.

[0083] The active compounds can also be in micro-encapsulated form withone or more excipients as noted above. The solid dosage forms oftablets, dragees, capsules, pills, and granules can be prepared withcoatings and shells such as enteric coatings, release controllingcoatings and other coatings well known in the pharmaceutical formulatingart. In such solid dosage forms the active compound may be admixed withat least one inert diluent such as sucrose, lactose or starch. Suchdosage forms may also comprise, as is normal practice, additionalsubstances other than inert diluents, e.g., tableting lubricants andother tableting aids such a magnesium stearate and microcrystallinecellulose. In the case of capsules, tablets and pills, the dosage formsmay also comprise buffering agents. They may optionally containopacifying agents and can also be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain part of theintestinal tract, optionally, in a delayed manner. Examples of embeddingcompositions which can be used include polymeric substances and waxes.

[0084] Dosage forms for topical or transdermal administration of acompound of this invention include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants or patches. The activecomponent is admixed under sterile conditions with a pharmaceuticallyacceptable carrier and any needed preservatives or buffers as may berequired.

[0085] Pharmaceutically acceptable diluents or carriers may be diluentsor carriers acceptable for topical application at the intended site oftherapy, e.g. diluents or carriers acceptable for topical administrationpulmonary, dermally, nasally, ocularly or rectaly.

[0086] The ointments, pastes, creams and gels may contain, in additionto an active compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

[0087] Powders and sprays can contain, in addition to the compounds ofthis invention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

[0088] Transdermal patches have the added advantage of providingcontrolled delivery of a compound to the body. Such dosage forms can bemade by dissolving or dispensing the compound in the proper medium.Absorption enhancers can also be used to increase the flux of thecompound across the skin. The rate can be controlled by either providinga rate controlling membrane or by dispersing the compound in a polymermatrix or gel.

[0089] Forms in topically administrable form, e.g. enabling orfacilitating topical administration, include, e.g. dry powderpreparations of the active ingredient (i.e. cyclosporin analog of theinvention) in substantially pure form, for example as employed in theart for delivery from a dry powder inhalation device. Means or devicesenabling or facilitating topical administration include, in particular,inhalation devices as well as containers and the like from which theactive ingredient may be delivered in a form capable of topicalapplication. Preferred embodiments as defined under C will be such aspermit topical administration within the airways or lungs, e.g. byinhalation.

[0090] It is clear that safety may be maximized by delivering the drugsby the inhaled route either in nebuliser form or as dry powder. Clearlythe great advantage of the inhaled route, over the systemic route, inthe treatment of asthma and other diseases of airflow obstruction and/orof chronic sinusititis, is that patients are exposed to very smallquantities of the drug and the compound is delivered directly to thesite of action.

[0091] Preparation of forms suitable for administration by inhalationmay be carried out by methods known in the art. It should be noted thatseveral antibiotics have recently developed for topical inhaled usage,particularly in cystic fibrosis, where they have been shown to beeffective against pseudomonas infections. Various inhalants aredescribed. For example, in DE 1491707, GB 1,392,945, GB 1,457,351, GB1,457,352, NL 147939, DE 1491715, GB 1,598,053, EP 5585, EP 41783, EP45419, EP 360463 and FR 2628638. DE 1491715, in particular, is said tobe suitable for inhalation therapy intended for bronchial or lungdiseases.

[0092] For this purpose cyclosporin analogs of the invention may beemployed in any suitable finely dispersed or finely dispersible form,capable of administration into the airways or lungs, for example infinely divided dry particulate form or in dispersion or solution in anyappropriate (i.e. pulmonarily administerable) solid or liquid carriermedium. For administration in dry particulate form, cyclosporin analogsof the invention may, for example, be employed as such, i.e. inmicronised form without any additive materials, in dilution with otherappropriate finely divided inert solid carrier or diluent (e.g. glucose,lactose, mannitol, sorbitol, ribose, mannose or xylose), in coatedparticulate form or in any other appropriate form as know in the art forthe pulmonary administration of finely divided solids.

[0093] Pulmonary administration may be effected using any appropriatesystem as known in the art for delivering drug substance in dry orliquid form by inhalation, e.g. an atomizer, nebulizer, dry-powderinhaler or like device. Preferably a metered delivery device, i.e.capable of delivering a pre-determined amount of cyclosporin analog ateach actuation, will be employed. Such devices are known in the art.

[0094] For nasal administration, cyclosporin analogs of the inventionwill suitably be administered in liquid form from a nasal applicator.Suitable topical forms for the treatment of diseases or conditions ofthe skin will include, for example, creams, gles, ointments, pastes,cataplasms, plasters, transdermal patches and the like. Formulations fordermal application will appropriately contain a skin penetrationenhancer, e.g. as know in the art, for example azone. Forms suitable forophthalmic use will include lotions, tinctures, gels, ointment andophthalmic inserts, again as known in the art. For rectaladministration, i.e. for topical therapy of the colon, cyclosporinanalogs of the invention may be administered in suppository or enemaform, in particular in solution, e.g. in vegetable oil or like oilysystem for use as a retention enema.

[0095] According to the present invention, cyclosporin analogs may beused for the manufacture of a topical preparation for the treatment,with or without the concurrent use of other drugs. For the abovepurposes, cyclosporin analogs of the invention may be employed in anydosage form appropriate for topical administration to the desired site.For example, for the treatment of diseases of the airways or lungs,cyclosporin analogs of the invention may be administered via thepulmonary route, by inhalation from an appropriate dispenser device.

[0096] Dosage for the topical preparation will in general be one tenthto one hundredth, of the dose required for oral preparation.Abbreviations Sar: Sarcosine MeLeu: N-Methyl-Leucine Val: Valine Ala:Alanine MeVal: N-Methyl Valine Et: Ethyl Ph: Phenyl Fmoc:9-Fluorenylmethoxycarbonyl- MeBmt:N-Methyl-(4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine α-Abu:α-Aminobutyric acid

[0097] Synthetic Methods

[0098] The compounds and processes of the present invention will bebetter understood, but are not limited to, the following syntheticscheme which illustrates the methods by which the compounds of thepresent invention (illustrated by formula I) may be prepared. The groupsX and Y, and the amino acid residues B and U in formula I are as definedearlier in the specification. The starting material for Scheme I,illustrated by formula I where A′=-MeBmt-, may be, for example, but notlimited to, a fermentation product or a synthetic product made bysolution phase chemistry. Preferably, the starting material iscommercially available. The starting material as a fermentation productmay be made from highly productive strains, for example, but not limitedto, Sesquicillopsis rosariensis G. ARNOLD F605; Tolypocladium inflatumwb6-5; Fusant, Tolypocladium inflatum KD461 etc. (in U.S. Pat. Nos.5,256,547; 5,856,141 etc.). Alternately, the starting material may bemade by solution phase chemistry either by sequentially assembling aminoacids or by linking suitable small peptide fragments, where the unitsare linked by, for example, but not limited to, amide, ester orhydroxylamine linkages (described in, Müller, Methoden der organischen,Chemie Vol. XV/2, pp 1 to 364, Thieme Verlag, Stuttgart, 1974; Stewart,Young, Solid Phase Peptide Synthesis, pp 31 to 34, 71 to 82, PierceChemical Company, Rockford, 1984; Bodanszky, Klausner, Ondetti, PeptideSynthesis, pp 85 to 128, John Wiley & Sons, New York, 1976 and otherstandard books on solution phase peptide chemistry). For amide linkagesparticular preference is given to the azide method, the symmetric andmixed anhydride method, in situ generated or preformed active esters andmethods using coupling reagents (e.g., dicyclohexylcarbodiimide,N,N-dimethyl-4-aminopyridine, N-hydroxy-benzotriazole, PyBrop® etc.).Classical solution phase chemistry using standard Z- and Boc-methodology may be used.

[0099] Residue A, which is -MeBmt- in the starting material is furthermodified, as illustrated in the following reaction scheme.

[0100] The process for the preparation of the compounds of formula Icomprises reacting a compound of formula I, where A′=-MeBmt- (forexample, Cyclosporin A) with an olefin having a terminal double bondwith catalysts such as Grubb's ruthenium alkylidene, Grubbsdihydroimidazole ruthenium, Shrock-Hoveyda molybdenum catalysts orbenzylidene catalysts [see (a) U.S. Pat. No. 6,111,121; (b) Reviews:Synlett, 1999, 2, 267; (c) Reviews: Ivin, K J; Mol, J. C. OlefinMetathesis and Metathesis Polymerization, 2^(nd) ed., Academic Press,New York, 1997; (d) J. Org. Chem., 1999, 64, 4798-4816; (e) Angew.Chem., Int. Ed. English, 1997, 36, 2036-2056; (f) Tetrahedron 1998, 54,4413-4450.] or Nolan's ruthenium catalyst [see (a) International PatentApplication No. WO 00/15339; (b) Org. Lett., 2000, 2, 1517-1519; (c) J.Org. Chem., 2000, 65, 2204-2207] or Molybdenum catalysts [see (a) J. Am.Chem. Soc., 1990, 112, 3875 (b), J. Am. Chem. Soc., 1996,118,10926-10927] in the presence of a lithium salt such as lithiumbromide, lithium chloride, lithium trifluoroacetate, lithium triflate ofa lewis acid such as titanium isopropoxide in an organic solvent. Theorganic solvent used may be solvents such as, for example,dichloromethane, chloroform, toluene, benzene, tetrahydrofuran,dimethylformamide and the like or mixtures thereof. The reaction may becarried out from room temperature to about 100° C. for 1-7 days toprovide a compound of formula I, where residue A′ is converted toresidue A″ having formula (i).

[0101] The compounds of formula I in an organic solvent, where residueA″ has formula (i), are then subjected to standard hydrogenationconditions using a catalyst such as, but are not limited to, a catalyticamount of palladium on carbon in a hydrogen atmosphere to provide thesaturated compounds of formula I, where in particular, residue A″ havingformula (i) is converted to residue A, as described throughout thespecification.

[0102] The organic solvents used can be solvents such as methanol,ethanol, ethyl acetate or mixtures thereof. Other catalysts useful toassist hydrogenation may be, for example, but not limited to, platinummetal or its oxide [see standard books on catalytic hydrogenation, e.g.,Rylander, P. N., Hydrogenation Methods, Academic Press: NY, 1985;Catalytic Hydrogenation in Organic Synthesis, Academic Press: NY, 1985;{haeck over (C)}ervený, L., Catalytic Hydrogenation, Elsevier: NY, 1986etc.]. The reaction may be carried out at room temperature or elevatedtemperature, for example, but not limited to, 50° C. or 100° C.

EXAMPLES

[0103] The procedures described above for preparing the compounds of thepresent invention will be better understood in connection with thefollowing examples, which are intentended to be illustrative only andnot limiting of the scope of the invention. Various changes andmodifications of the disclosed embodiments will be apparent to thoseskilled in the art. Such changes and modifications, including withoutlimitation, those relating to the chemical structures, substituents,derivatives, intermediates, syntheses, formulations and/or methods forthe invention may be made without departing from the spirit of theinvention and the scope of the appended claims.

Example 1 Compound of Formula I, Where in Residue A, X is Absent andY=—COOCH₃, Residue, B=-αAbu-, and Residue, U=-(D)Ala-

[0104] Cyclosporin methyl ester (0.030 mg, 0.024 mmol) and palladium oncarbon (0.0012 mg, 0.0012 mmol) were added to a flask and the flask wasevacuated and backfilled with hydrogen gas three times. Anhydrousmethanol (3 ml) was added and the reaction was stirred for 18 h atambient temperature under an atmosphere of hydrogen. After filtrationand concentration in vacuo, the product was isolated as a white solid(0.021 mg, 70 % yield). Electrospray mass spectrum (ESMS) M+H: 1248.91

Example 2 Compound of Formula I, Where in Residue A, X is Absent andY=—COOEt; Residue B=-αAbu-, and Residue U=-(D)Ala-

[0105] The title compound of example 2 was prepared from cyclosporinethyl ester and palladium on carbon according to the proceduresdescribed in Example 1. ESMS M+H: 1262.3

Example 3 Compound of Formula I, Where in Residue A, X is Absent andY=—COOCH₂CH₂CH₃; Residue B=-αAbu-, and Residue U=-(D)Ala-

[0106] The title compound of example 3 was prepared from cyclosporinpropyl ester and palladium on carbon according to the proceduresdescribed in Example 1.

Example 4 Compound of Formula I, Where in Residue A, X is Absent andY=—COOCH₂Ph; Residue B=-αAbu-, and Residue U=-(D)Ala-

[0107] The title compound of example 4 was prepared from cyclosporinbenzyl ester and palladium on carbon according to the proceduresdescribed in Example 1.

Example 5 Compound of Formula I, Where in Residue A, X is absent andY=—COOCH₂F; Residue B=-αAbu-, and Residue U=-(D)Ala-

[0108] The title compound of example 5 was prepared from cyclosporinfluoromethyl ester ester and palladium on carbon according to theprocedures described in Example 1

Example 6 Compound of Formula I, Where in Residue A, X is Absent andY=—COOCHF₂; Residue B=-αAbu-, and Residue U=-(D)Ala-

[0109] The title compound of example 6 was prepared from cyclosporindifluoromethyl ester ester and palladium on carbon according to theprocedures described in Example1

Example 7 Compound of Formula I, Where in Residue A, X is Absent andY=—COOCF₃; Residue B=-αAbu-, and Residue U=-(D)Ala-

[0110] The title compound of example 7 was prepared from cyclosporintrifluoromethyl ester ester and palladium on carbon according to theprocedures described in Example 1.

Example 8 Compound of Formula I, Where in Residue A, X is Absent andY=—COOCH₂CF₃; Residue B=-αAbu-, and Residue U=-(D)Ala-

[0111] The title compound of example 8 was prepared from cyclosporintrifluoroethyl ester ester and palladium on carbon according to theprocedures described in Example 1.

[0112] The cyclosporin analogs of the present invention have potentimmunosuppressive and anti-inflammatory activity. In particular, theyinhibit antigen-induced inflammatory cell infiltration, for example,into the airways. In vivo this activity is apparent following topicaladministration, e.g., pulmonary route.

[0113] The immunosuppressive and anti-inflammatory properties ofcyclosporin analogs of the invention may be demonstrated in standardtest models in vitro and in vivo for example as follows.

Example 9 Calcineurin Inhibition Assay

[0114] The immunosuppressive activity of cyclosporin is mediated throughinhibition of the phosphatase activity of the enzyme calcineurin by acyclophilin-cyclosporin complex. Thus, calcineurin inhibition is widelyused as an in vitro measure of the activity of cyclosporin analogs.

[0115] Compounds were tested in an assay based on the Biomol GreenCalcineurin Assay Kit supplied by Biomol (Plymouth Meeting, Pa.),supplemented with Cyclophilin A for enzyme inhibition. The activity ofthe recombinant human calcineurin was determined by release of phosphatefrom a phosphopeptide representing a fragment of camp-dependent proteinkinase. Phosphate release was determined using the calorimetricdetection reagent Biomol Green (Biomol AK-111).

[0116] Compounds in DMSO (2.4 μl) were added to a 96-well microplate andmixed with 50 μl assay buffer (50 mM Tris-HCl, pH 7.5; 100 mM sodiumchloride; 6 mM magnesium chloride; 0.5 mM dithiothreitol, 0.025% NP-40,500 μM calcium chloride, 0.27 μM Calmodulin) containing 10 μMCyclophilin and 3 nM Calcineurin. After warming to 37° C. for 60 mins,the enzymatic reaction was initiated by addition of phosphopeptide (7.5μl) to give a final concentration of 94 μM. Phosphate release after 60min at 37° C. was determined by addition of Biomol Green (100 μl) andmeasurement of the absorbance at 620 nm after 15 mins at roomtemperature.

[0117] IC₅₀ values were calculated from determinations of enzymeactivity at inhibitor concentrations ranging from 0.1 to 0.0015 μM.

Example 10 NFAT Reporter Gene Assay

[0118] NFAT activation follows precisely the activation of calcineurinby increased free calcium levels in the cytoplasm. Researchers fromdiverse fields are interested in the NFAT family of transcriptionfactors, which are potential targets for newer and saferimmunosuppressive drugs. In addition, the activation of NFAT proteinsinvolves various cellular signal transduction pathways, includingcalcium mobilization and MAP kinase pathways linked to T-cell receptorsand Ras1. To assist researchers probing the activity of NFAT proteins,Stratagene has developed a PathDetect cis-reporter plasmid, thepNFAT-Luc reporter plasmid (Stratagene, Inc. catalog #219094),containing the NFAT binding site from the human IL-2 gene.2,7-9. TheNFAT cis-reporting system includes the transfection-ready pNFAT-Lucreporter plasmid and the pCIS-CK negative control plasmid.

[0119] Construction of the pNFAT-Luc Plasmid:

[0120] The backbone of the 5749-base-pair pNFAT-Luc plasmid is thepFR-Luc reporter plasmid of the aforementioned PathDetecttrans-reporting system. To this backbone, the GAL4 binding element wasreplaced with four direct repeats of the NFAT binding sequence (−286 to−257) from the IL-2 gene promoter, the most studied and widely used NFATbinding sequence. For all reporter plasmids of the PathDetectcis-reporting systems, activation of the luciferase gene indicatedinteraction of uncharacterized gene products, extracellular stimuli,growth factors, or drug candidates with specific enhancer elements. Thena plasmid expressing the gene of interest was cotransfected intomammalian cells along with a cis-reporter plasmid to indicatetranscription activation.

[0121] Testing the pNFAT-Luc Plasmid in Jurkat Cells:

[0122] Pharmacology studies have established that NFAT proteins can beactivated by the protein kinase C activator phorbol ester (PMA) incombination with the calcium ionophore ionomycin, reagents that raisefree intracellular calcium. When Jurkat cells, a mature human T-cellline, or CHO cells were transfected with the pNFAT-Luc plasmid andtreated with 60 ng/ml of PMA and 1 μg/ml of inomycin, luciferaseactivity increased by 13- and 16-fold, respectively. Therefore, theenhancer element in the pNFAT-Luc plasmid is responsive to calciummobilization. Cells transfected with pNFAT-Luc and then treated witheither PMA or ionomycin alone did not show a significant increase inluciferase activity.

[0123] Cyclosporin inhibits the activity of calcineurin, a proteinphosphatase regulated by intracellular calcium mobilization. All theisoforms of NFAT protein contain a calcineurin-binding domain and areactivated by calcineurin. The inhibition of luciferase expression frompNFAT-Luc in the present model, in both Jurkat and CHO cells induced byPMA and ionomycin, was monitored for cyclosporin (as a positive control)and the cyclosporin analogs of the present invention.

[0124] In another set of experiments, rat basophilic leukemia cellsstably transfected with chemokine receptors were transtected withpNFAT-Luc and then treated with their respective ligands (data notshown). When both luciferase expression and calcium levels weremonitored in these cells, luciferase expression correlated very wellwith calcium mobilization. Therefore, luciferase expression frompNFAT-Luc indeed reflects the activation of endogenous NFAT proteins bycalcium immobilization.

Example 11 Immunosuppressive Activity and Applications Murine MixedLymphocyte Reaction

[0125] Ca. 0.5×10⁶ lymphocytes from the spleen of female (8-10 weeks)Balb/c mice are incubated for 5 days in 0.2 ml cell growth medium withca. 0.5×10⁶ lymphocytes from the spleen of female (8-10 weeks) CBA mice.Test substance is added to the medium at various concentrations.Activity is assessed by ability to suppress proliferation-associated DNAsynthesis as determined by incorporation of radiolabelled thymidine.

Mishell-Dutton Test

[0126] Ca. 10⁷ lymphocytes from the spleen of OFI, female mice areco-cultured with ca. 3×10⁷ sheep erythrocytes for 3 days. Test substanceis added to the incubation medium in varying concentrations. Lymphocytesare harvested and plated onto agar with fresh sheep erythrocytes asantigen. Sensitized lymphocytes secrete antibody that coats theerythrocytes, which lyse to form a plaque in the presence of complement.Activity is assessed by reduction in the number of plaque forming, i.e.,antibody product, cells.

Delayed-type Hypersensitivity Resonse

[0127] On Day 0 groups of ten mice (having BALB/cByJ or any otheracceptable strain) are dosed with test compound (1 to 10%), vehicle orthe positive control, cyclophosphamide (Cyclosporin A), and monitoredfrom Day-2 to 7. The mice are anesthetized and their abdomens shaved.100 μl of a 3% solution of ovalbumin are applied to the abdomen anddried. Seven days later, the mice are challenged by applying 5 μl ofovalbumin to each side of the right ear. After 24 hours, both the rightand left ear thickness are measured using a micrometer caliper.

Popliteal Lymph Node Assay

[0128] First, an inducer (phenytoin) is injected into the mice footpad(having BALB/cByJ or any other acceptable strain). Then the mice arechallenged (subcutaneously or po) with ester and control agent usinggraded doses, for example, 2.5, 10, 20 mg/Kg (based on cyclosporine Adata). On day 7 the popliteal lymph nodes are excised from the dosedmice and the lymph nodes are weighed. Then single cell suspensions ofeach lymph node are prepared and enumerated. The weight index for eachanimal is calculated (for example, a mean weight index<2 would indicatesuppression of immune response).

Influence on Allergen-induced Pulmonary Eosinophilia (in vitro)

[0129] Male Himalayan spotted guinea pigs (300 g, BRL) are sensitized toovalbumin (OA) by i.p. injection of 1 ml of a suspension of OA (10μg/ml) with Al(OH)₃ (100 mg) and B-pertussis vaccine (0.25 ml) in saline(0.9% w/v). For oral studies, the procedure is repeated 1× after 2 weeksand the animals are used one week later. For inhalation studies, theprocedure is repeated 2× at 3-week intervals and the animals are usedone week after the last injection.

[0130] Challenge is effected employing a saline solution of OA,nebulized for discharge into an exposure chamber. Test animals areexposed to OA by nose only inhalation for 60 minutes. For inhalationstudies, OA solution is used at a concentration of 0.01%.

[0131] Test substance is administered (a) inhalation and/or (b) orally.For oral studies, test substance is administered p.o. in olive oil 1×daily for 3 days or in powder form in methylcellulose once prior to OAchallenge. On day 3, test animals receive test substance 1.5 hrs. priorto and 6 hrs. after OA challenge. For inhalation studies, testsubstanceis micronised for delivery to test animals restrained within aflow-past, nose-only inhalation chamber. Administration by inhalation iseffected 15 mins. prior to OA challenge.

[0132] Efficacy of administered test substance is determined bybronchoalveolar lavage (BAL) and cell counting. For this purpose animalsare sacrificed with Na pento-barbitone (100 mg/kg i.p.) and the tracheais exposed and cannulated. 5 successive 10 ml aliqots of Ca²+ and Mg²+free Hank's balanced salt solution (HBSS), containing bovine serumalbumin (BSA, 0.3%), EDTA (10 mM) and HEPES (10 mM) is then introducedinto the lung and immediately aspirated by gentle compression of thelung tissue. Total cell counts in pooled eluates are determined using anautomatic cell counter. Lavage fluid is centrifuged at 200 g for 10minutes and the cell pellet resuspended in 1 ml of supplemented HBSS. 10μl of this cell suspension is added to 190 μl of Turk's solution (1:20)dilution). Differential cell counts are made from smears stained byDiff-Quick. Cells are identified and counted under oil immersion(×1,000). A minimum of 500 cells per smear are counted and the totalpopulation of each cell type is calculated.

[0133] In untreated animals, OA challenge induces increase of all celltypes in BAL fluid 24 hours after challenge. Prior administration ofcyclosporin analogs in accordance with the present invention byinhalation at dosages of the order of from 1.0 to 15.0 mg/kg reduceseosinophil count in BAL in a dose dependent manner as compared withuntreated controls. Cell counts for other leucocytes (macrophages,neutrophils etc.) are also reduced.

What is claimed is:
 1. A cyclosporin analog of formula (I) or a pro-drugor a pharmaceutically acceptable salt thereof:

wherein, (a) A is of the formula:

wherein X is absent, —C1-C6 alkyl-, or —C3-C6 cycloalkyl-; Y is selectedfrom the group consisting of: i. —C(O)—O—R1 where R1 is hydrogen, C1-C6alkyl optionally substituted with halogen, heterocyclics, aryl, C1-C6alkoxy or C1-C6 alkylthio, halogen substituted C1-C6 alkoxy, halogensubstituted C1-C6 alkylthio; ii. —C(O)—S—R1 where R1 is hydrogen, C1-C6alkyl optionally substituted with halogen, heterocyclics, aryl, C1-C6alkoxy or C1-C6 alkylthio, halogen substituted C1-C6 alkoxy, halogensubstituted C1-C6 alkylthio; iii. —C(O)—OCH2-OC(O)R2 where R2 is C1-C6alkyl, optionally substituted with halogen, C1-C6 alkoxy, C1-C6alkylthio, heterocyclics or aryl; iv. —C(S)—O—R1 where R1 is hydrogen,C1-C6 alkyl optionally substituted with halogen, heterocyclics, aryl,C1-C6 alkoxy or C1-C6 alkylthio, halogen substituted C1-C6 alkoxy,halogen substituted C1-C6 alkylthio; and v. C(S)—S—R1 where R1 ishydrogen, C1-C6 alkyl optionally substituted with halogen,heterocyclics, aryl, C1-C6 alkoxy or C1-C6 alkylthio, halogensubstituted C1-C6 alkoxy, halogen substituted C1-C6 alkylthio. (b) B is-αAbu-, -Val-, -Thr- or -Nva-; and (c) U is -(D)Ala-, -(D)Ser- or-[O-(2-hydroxyethyl)(D)Ser]-; or -[O-acyl(D)Ser]- or-[O-(2-acyloxyethyl)(D)Ser]-.
 2. A cyclosporin analog according to claim1 or a pro-drug or a pharmaceutically acceptable salt thereof, whereinin formula (I), B is -αAbu-, and U is -(D)Ala-.
 3. A cyclosporin analogaccording to claim 1 or a pro-drug or a pharmaceutically acceptable saltthereof, wherein in formula I: (i) A is of the formula A1 or A2,wherein: X is absent; and Y is selected from a group consisting of: i.—C(O)—O—R1 where R1 is hydrogen, C1-C6 alkyl optionally substituted withhalogen, heterocyclics, aryl, C1-C6 alkoxy or C1-C6 alkylthio, halogensubstituted C1-C6 alkoxy, halogen substituted C1-C6 alkylthio; ii.—C(O)—S—R1 where R1 is hydrogen, C1-C6 alkyl optionally substituted withhalogen, heterocyclics, aryl, C1-C6 alkoxy or C1-C6 alkylthio, halogensubstituted C1-C6 alkoxy, halogen substituted C1-C6 alkylthio; and iii.C(O)—OCH₂—OC(O)R2 where R2 is C1-C6 alkyl optionally substituted withhalogen, C1-C6 alkoxy, C1-C6, alkylthio, heterocyclics or aryl; (ii) Bis -αAbu-; and (iii) U is -(D)Ala-.
 4. A cyclosporin analog according toclaim 1 or a pro-drug or a pharmaceutically acceptable salt thereof,selected from the group consisting of: Compound of Formula (I) whereinB=-αAbu-, U=-(D)Ala-, X is absent, Y=—COOCH₃; Compound of Formula (I)wherein B=-αAbu-, U=-(D)Ala-, X is absent, Y=—COOH; Compound of Formula(I) wherein B=-αAbu-, U=-(D)Ala-, X is absent, Y=—COOEt; Compound ofFormula (I) wherein B=-αAbu-, U=-(D)Ala-, X is absent, Y=—COOCH₂CH₂CH₃;Compound of Formula (I) wherein B=-αAbu-, U=-(D)Ala-, X is absent,Y=—COOCH₂Ph; Compound of Formula (I) wherein B=-αAbu-, U=-(D)Ala-, X isabsent, Y=—COOCH₂F; Compound of Formula (I) wherein B=-αAbu-,U=-(D)Ala-, X is absent, Y=—COOCHF₂; Compound of Formula (I) whereinB=-αAbu-, U=-(D)Ala-, X is absent, Y=—COOCF₃; Compound of Formula (I)wherein B=-αAbu-, U=-(D)Ala-, X is absent, Y=—COOCH₂CF₃; Compound ofFormula (I) wherein B=-αAbu-, U=-(D)Ala-, X is absent, Y=—COOCH₂Cl;Compound of Formula (I) wherein B=-αAbu-, U=-(D)Ala-, X is absent,Y=—COOCH₂OCH₃; Compound of Formula (I) wherein B=-αAbu-, U=-(D)Ala-, Xis absent, Y=—COOCH₂OCH₂CH₂OCH₃; Compound of Formula (I) whereinB=-αAbu-, U=-(D)Ala-, X is absent, Y=—C(═O)SCH₂Ph; Compound of Formula(I) wherein B=-αAbu-, U=-(D)Ala-, X is —CH₂CH₂CH₂—, Y=—COOCH₃; andCompound of Formula (I) wherein B=-αAbu-, U=-(D)Ala-, X is absent,Y=—COOFmoc.
 5. A chemical process for preparing a cyclosporin analog offormula I as claimed in claim 1, comprising: a. reacting a compound offormula I, wherein A=-MeBmt- with: i. an olefin of formula CH₂═CH—X—Y,wherein X and Y are as defined in claim 1; and ii. a catalyst; in thepresence of a lithium salt in an organic solvent; and b. hydrogenatingthe product of step a in an organic solvent under hydrogen with acatalyst; and optionally converting the product of said reaction into apharmaceutically acceptable salt.
 6. The chemical process as claimed inclaim 5, wherein the catalyst in step (a) (ii) is Grubb's rutheniumalkylidene, Nolan's catalyst, a benzylidene catalyst or a molybdenumcatalyst.
 7. The chemical process as claimed in claim 5, wherein step(b) is performed at room temperature.
 8. The chemical process as claimedin claim 7, wherein the catalyst in step (b) is Palladium on carbon. 9.A pharmaceutical composition, said composition comprising at least onecyclosporin analog of formula 1 as claimed in claim 1, said cyclosporinanalog being present alone or in combination with a pharmaceuticallyacceptable carrier or excipient.
 10. A method for treating diseasescharacterized by airflow obstruction in a subject in need of treatmentwhich comprises the step of administering to said subject atherapeutically effective amount of at least one cyclosporin analog offormula I as claimed in claim
 1. 11. The method of claim 10, whereinsaid disease is asthma.
 12. The method of claim 10, wherein the step ofadministering the cyclosporin analog of formula I is done by topicaladministration.